Dilated cardiomyopathy (DCM) is the third most common cause of heart failure, often associated with arrhythmias and sudden cardiac death if not controlled. Metabolic and/or environmental factors, such as alcohol abuse, obesity, diabetes and Chagas disease, alter glycoprotein glycosylation, can lead to DCM. Inherited genetic disease, such as the human congenital disorders of glycosylation (CDG), causes multi-system manifestations including DCM. Non-congenital changes in glycosylation are also occurred in humans with and in animal models of DCM and heart failure. However, mechanisms responsible for glyco-dependent DCM are not understood. Here we sought to investigate the impact of sialylation and N-glycosylation in cardiac function. Partial reduction of N-α2,3-sialylation achieved through ST3Gal4 deletion (ST3Gal4-/-) led to adult late-onset DCM. The DCM symptoms progressed gradually, developing thinner left ventricular walls and dilation of all four chambers by 18-month old, but with preserved systolic function. Transverse aortic constriction (TAC) was used as a chronic stressor on 16-20 week old mice to determine whether the ability of the ST3Gal4-/- heart to compensate against pathologic insult is compromised. TAC’d ST3Gal4-/- mice presented with insufficient hypertrophy and reduced systolic function that deteriorated into congestive HF within six weeks post-surgery, while constricted WT hearts remained well-adapted throughout (ejection fraction, ST3Gal4-/-=34±5.2%; WT =53.8±7.4%; p<0.05).Calcineurin expression was decreased in ST3Gal4-/- (compared to TAC’d WT), contributed to the maladaptation of TAC’d ST3Gal4-/-. In order to better understand the role of glycosylation on cardiac function, we generated a cardiomyocyte specific knockout (αMHC-Cre) of glycosyltransferase responsible for synthesizing complex and hybrid N-glycans, Mgat1, (Mgat1CKO). Similar to but much more severe than that observed in ST3Gal4-/-, Mgat1CKO developed early-onset of DCM, late adult mortality, severely impaired cardiac systolic and diastolic function and frequent arrhythmias. Marked sex-difference in cardiac phenotype was observed in this autosomal gene (Mgat1) deletion, with male Mgat1CKO more severely affected. Both ST3Gal4 and Mgat1 did not participate in murine cardiogenesis, evidenced by normal litter size, Mendelian distribution of genotypes, no septal defect or vessel deformation under autopsy or echocardiography. In conclusion, we provided here the first and direct evidence of desialylation-elicited idiopathic dilated cardiomyopathy (DCM), reporting the cardiac phenotype of ST3Gal4-/-and cardiac-specific knockout of Mgat1. Our data showed sialylation and complex N-glycosylation are essential for cardiac function, and reduced N-glycosylation or sialylation leads to DCM development, contractile dysfunction and arrhythmia.

Congenital disorders of glycosylation (CDG) represent a rapidly growing group of rare inherited metabolic diseases with estimated prevalence as high as 1:20 000, which are caused by genetic defects that impair the process of glycosylation, i.e. the enzymatic addition of a specific saccharide structure onto a protein or lipid backbone. Due to non-specificity and variability of clinical symptoms in the patients, the medical diagnosis of CDG remains extremely challenging and significantly relies on accurate biochemical and genetic analyses. The overall goal of the present dissertation thesis was to study CDG at the biochemical and molecular genetic level in the context of the Czech and Slovak Republic, which involved three specific aims: A.) to introduce and optimize laboratory screening methods for CDG detection in a group of clinically suspected patients, B.) to determine the corresponding genetic defect in the positive patients selected via CDG screening and to study the pathobiochemical aspects of specific CDG types at the cellular level, and C.) to analyze glycosylation disturbances of non- CDG etiology. Contributions of this work include optimization of isoelectric focusing of apolipoprotein C-III (ApoC-III) as a screening method for O-glycosylation abnormalities, as well as the description of...

Dolichol is a membrane lipid, which carries monnosaccharides and glycans for N-linked protein glycosylation and glycosylphosphatidylinositol-anchor biosynthesis occuring in endoplasmic reticulum. Its structure is composed of isoprenoid units. Dolichol is present in all tissues and in most of the membrane organelles of eukaryotic cells. Recently some types of congenital disorders of glycosylation have been described as a consequence of dolichol biosynthesis and metabolism defects, which are not detectable by standard methods. The aim of this diploma thesis was to analyze dolichol content in urine and in different tissues from patients with deficiency in dolichol biosynthesis by mass spectrometry and to study the impact of these defects on energetic metabolism. Biological material for this study consisted of urine samples from 76 controls with age ranging from 1 months to 81 years, 6 patients with congenital disorders of glycosylation and 43 patients with suspicion of congenital disorder of glycosylation; samples of frontal cortex, liver, muscle and heart tissues from 2 patients with mutation in NUS1 gene and controls. Urine samples were stored at -20 řC and tissue homogenates were stored in -80 řC until analysis. Lipid fraction after extraction was separated by liquid chromatography. Dolichols were...

Congenital disorders of glycosylation (CDG) represent a rapidly growing group of rare inherited metabolic diseases with estimated prevalence as high as 1:20 000, which are caused by genetic defects that impair the process of glycosylation, i.e. the enzymatic addition of a specific saccharide structure onto a protein or lipid backbone. Due to non-specificity and variability of clinical symptoms in the patients, the medical diagnosis of CDG remains extremely challenging and significantly relies on accurate biochemical and genetic analyses. The overall goal of the present dissertation thesis was to study CDG at the biochemical and molecular genetic level in the context of the Czech and Slovak Republic, which involved three specific aims: A.) to introduce and optimize laboratory screening methods for CDG detection in a group of clinically suspected patients, B.) to determine the corresponding genetic defect in the positive patients selected via CDG screening and to study the pathobiochemical aspects of specific CDG types at the cellular level, and C.) to analyze glycosylation disturbances of non- CDG etiology. Contributions of this work include optimization of isoelectric focusing of apolipoprotein C-III (ApoC-III) as a screening method for O-glycosylation abnormalities, as well as the description of...

CDG-Ia ist eine genetisch bedingte Stoffwechselstörung, die eine fehlerhafte Synthese von Glykoproteinen zur Folge hat. Ursache sind Mutationen in der genetischen Information für das Enzym Phosphomannomutase II (PMM2). Inhalt dieser Arbeit ist die Generierung von zwei Gene-Targeting-Vektoren (GTV) und den entsprechenden transgenen embryonalen Maus-Stammzellen, welche jeweils die beiden häufigsten Mutationen R141H und F119L (R137H und F115L im Maus-Genom) tragen. Ausgangsprodukt ist ein Teil des murinen Pmm2-Gens, welcher in einen pBlueScript-Vektor subkloniert wird. Mit gezielter in-vitro-Mutagenese erfolgt das Einbringen der jeweiligen Mutation. Als Selektionsmarker für beide GTV dient ein von zwei loxP- Sequenzen flankiertes Neomycin-Resistenz-Gen (Neo). Die beiden derart konstruierten GTV, welche jeweils die Mutation F115L bzw. R137H beinhalten, werden als linearisiertes Konstrukt durch Elektroporation in embryonale Mausstammzellen transferiert. Nach Überprüfung der erfolgreichen homologen Rekombination wird je ein Stammzell-Klon mit dem Genotyp F115L/WT und R137H/WT mit einem Cre-Rekombinase-Gen transfiziert und die erfolgreiche Entfernung des Neo-Gens überprüft. Mit diesen transgenen Stammzellen sollen hypomorphe Mausmodelle für CDG-Ia generiert werden.

Congenital disorders of glycosylation (CDG) are rare multisystem metabolic diseases and their number has rapidly grown in recent years. The clinical manifestation includes very broad spectrum of symptoms. In most of all cases CDG are caused by mutations in genes encoding the enzymes of glycosylation pathway. Based on the type of defect, CDG are divided into the following groups: disorders of N-glycosylation or O-glycosylation of proteins, defects in modification of proteins by GPI anchor, disorders of lipid glycosylation and defects that impact multiple glycosylation pathways. The aim of the thesis was to find new biochemical analyses suitable for diagnostics and characterization of CDG patients. The experimental conditions were optimized for selected markers (Sambucus Nigra (SNA) lectin, proaerolysin (FLAER), antibodies to proteins CD55 and CD59) and the staining was applied to cultivated skin fibroblasts from controls and patients diagnosed with CDG by whole-exome sequencing (ATP6AP1-CDG, PIGN-CDG, SLC10A7-CDG, PISD deficiency). The experiments were performed using flow cytometry (FACS) and fluorescent microscopy (FM). The detection of sialylation by SNA lectin and analysis of the mitochondrial membrane potential changes by a fluorescent labelled probe JC-1 with FCCP simulation of mitochondrial...

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Glycolipids play important roles in the biology of prokaryotes and eukaryotes, including humans, and although theyare found on the cell-membrane surface of all eukaryotic cells, not much is known about their biosynthesis. The aim ofthis project was to characterize two enzymes: glucosylceramide synthase (GCS) which is involved in the biosynthesisof glycolipids such as gangliosides that are abundant in the membranes of nerve cells; and dolicholphosphate mannosesynthase (DPMS), involved in the synthesis precursor for protein glycosylation. Both GCS and DPMS have been shown play a role in cancer as well as in congenital disorders of glycosylation, and are therefore interesting targets tostudy from a therapeutic perspective.With the goal to identify a suitable expression system for GCS, the genes coding for GCS from lancelet (Branchiostoma floridae) and human (Homo sapiens) were cloned and tested for expression in Escherichia coliBL21(DE3)T1 and C41(DE3) using different vectors. Cloning into three different vectors was successful and initial expression testing was performed. SDS-PAGE analysis confirmed initial expression of proteins. Although the correctsize of the protein could be confirmed by Western blot, no fluorescence of the GFP-fusion protein could be detected.DPMS from Thermococcus onnurineus (ToDP) was expressed in E. coli C41(DE3) and purified by immobilized metal ion affinity chromatography and gel filtration. Crystallization optimization was performed for ToDP produced from the vector pNIC28-Bsa4 and plate-like crystals were obtained. X-ray intensity data analysis indicated that thesecrystals contained lipid rather than protein. Crystallization screening for ToDP produced from the vector pNIC-CTHO construct was successful. Crystallization screening using the commercially available MemGold-HT96 crystallization kit resulted in initial crystallization that yielded protein crystals that diffracted to 10 °A resolution. / Glykolipider är viktiga biologiska byggstenar hos prokaryoter och eukaryoter, även människor. Trots att glykolipider finns på cellmembran ytan hos alla eukaryota celler är inte mycket känt kring syntesen av glykolipider. Målet med detta projekt var att karaktärisera två enzym: glukosylceramidsyntas (GCS) som är involverat i biosyntesen av glykolipider som gangliosider vilka förekommer i cellmembranet hos människors nervceller; och dolikolfosfatmannossyntas (DPMS) som är involverat i syntesen av substrat för proteinglykosylering. Både GCS och DPMS harvisat sig spela en roll i cancer och medfödda glykosyleringssjukdomar och är därför intressanta enzym att studera ur ett medicinskt perspektiv.Med målet att identifiera ett lämpligt expressionssystem för GCS, klonades gener från lansett (Branschiostomafloridae) och människa (Homo sapiens) och testades för expression i Escherichia coli BL21(DE3)T1 och C41(DE3)med olika vektorer. Kloning av tre olika vektorer lyckades och expressionstester utfördes. Analys med SDS-PAGE bekräftade expression av protein. Trots att korrekt storlek av proteinet kunde bekräftas med Western blot, detekterades ingen fluorescens från GFP-fusionsproteinet. DPMS från Thermococcus onnurineus (ToDP) i två olika konstrukt uttrycktes i E. coli C41(DE3) och renades med immobiliserad metalljonaffinitetskromatografi och gelfiltrering. Kristalliseringsoptimering utfördes för ToDP uttryckt i vektorn pNIC28-Bsa4 och skivliknande kristaller erhölls. Diffraktionsdata indikerade dock att kristallerna innehöll lipider och inte protein. Kristallisering av ToDP uttryckt i vektorn pNIC-CTHO lyckades och initiala kristallingsförhållanden hittades genom att använda det kommersiellt tillgängliga kristalliseringskitet MemGold-HT96. Diffraktionsdata visade på upplösning ner till 10 Å.